Previously, viable chimeric flaviviruses were constructed that contained tick-borne encephalitis virus (TBEV) structural protein CME or ME genes with the remaining genes derived from dengue type 4 virus (DEN4). The ME chimera retained the neurovirulence for mice of its TBEV parent from which its M and E genes were derived, but it lacked the peripheral invasiveness of TBEV. The ME chimera was subjected to mutational analysis in an attempt to reduce or ablate neurovirulence manifest when virus was inoculated directly into the brain. Three distinct mutations were independently associated with marked reduction of mouse neurovirulence. These mutations ablated: (i) the TBEV PreM cleavage site which is required for proper processing of M protein; (ii) the TBEV E (envelope glycoprotein) glycosylation site; or (iii) the first DEN4 NS1 (non-structural protein one) glycosylation site. Each of the three attenuated mutants was restricted in growth in both simian and mosquito cells. Significantly, parenteral inoculation of these attenuated mutants induced complete resistance in mice to fatal encephalitis caused by subsequent challenge with the highly neurovirulent ME chimera. These observations suggest a new strategy for developing a live attenuated TBEV vaccine. Unlike the highly virulent TBEV, the wild type Langat virus (TP21 strain), the least virulent of all TBEV-complex flaviviruses, has low encephalitogenic potential and peripheral virulence and has not been reported to be associated with any human disease. In an attempt to identify the molecular basis for attenuation of TBEV-complex viruses, the sequence of the genome of wild type virus (TP21 strain) and a more attenuated strain of LGT derived from it (strain ES) were determined. Among the tick-borne flaviviruses, the LGT genome differs in length of its 3' noncoding region compared to TBEV or Powassan virus (a TBE virus of North America) genome. Analysis of the TP21 and E5 genome sequences revealed six amino acid differences in the polyprotein, one of them is a substitution Asn > Asp in position 387 of E protein, which probably is responsible for attenuation of E5 virus. Attenuating mutations which were identified by genetic analysis of the LGT strains will be introduced into chimeric LGT/DEN4 and TBEV/DEN4 genomes and progeny viruses will be analyzed for immunogenicity and loss of virulence.